The Markey Scholars Conference Proceedings (2004) / Chapter Skim
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11 Abstracts of Poster Sessions
11 Abstracts of Poster Sessions
Pages 97-152
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Abstracts of Poster Sessions
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Department of Medicine, Biochemistry and Molecular Biology Mount Sinai School of Medicine Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. "Blood islands" of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper (epiblast)
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As expected, downstream targets of the Hh signaling pathway (Ptch1, Smo, Gli1) are upregulated in anterior epiblasts cultured in the presence of Ihh protein.
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. Fortunately, as modern biomedical research methodologies evolved, these "arcane" fluorescence spectroscopic techniques became some of the key detection technologies associated with much of the modern biomedical research "revolution." Fluorescence polarization /anisotropy spectroscopy has become an essential component of high throughput homogenous drug screening and protein-protein interaction assays.
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To determine if transcription of this gene is regulated by such a complex, we analyzed the contributions of TAL1 and GATA-1 to Protein 4.2 DNA binding activity, promoter activity, and endogenous gene expression and in vivo occupancy of the Protein 4.2 promoter by TAL1. First, several TAL1-, GATA1-, LMO2-, and Ldb1-containing complexes were detected by gel mobility shift analysis of erythroid cell extracts using probes corresponding to either E box-GATA element in the Protein 4.2 promoter.
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Finally, an increase in endogenous Protein 4.2 gene expression and in E box-GATA DNA-binding activities was observed when TAL1 was overexpressed in MEL cells, a decrease in both was observed with enforced expression of a TAL1 mutant defective in DNAbinding or an Ldb1 mutant impaired in dimerization, and evidence for in vivo occupancy of the Protein 4.2 promoter by TAL1 was obtained through chromatin immunoprecipitation analysis. In sum, these data establish the Protein 4.2 gene as a direct target of a TAL1- and GATA-1-containing DNA-binding complex in differentiating erythroid cells.
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Single-minded and the control of CNS midline cell development. The CNS midline cells comprise a distinct set of functional neurons and glia, and also act as a signaling center that controls aspects of axon guidance, cell migration, and formation of epidermal, mesodermal, and neural cell types.
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Current work is focused on identifying genes that mediate CNS midline cell fate development, and understanding how Single-minded: Tango interacts with additional regulatory proteins to control their expression. PAS Proteins.
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We have shown that the crustacean, Artemia, has a trachealess ortholog that is prominently expressed in the epipodal gills. Although the insect trachea and crustacean gills are divergent morphologically, these results suggest that their formation may be controlled by the same regulatory gene.
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HIJACKING THE RIBOSOME: STRUCTURAL BASIS FOR TRANSLATION INITIATION IN HEPATITIS C VIRUS Jennifer A Doudna, Ph.D.
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One project focuses on how Pseudomonas aeruginosa (PA) , an important opportunistic pathogen of man, injures epithelial cells.
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Using confluent MDCK cells as a model system for an epithelial monolayer, we have shown that the differentiation state of the monolayer affects the ability of PA to damage or enter epithelial cells. ExoU is a novel cytotoxin that is directly translocated by the type III secretion system from the bacterium to the host cell where it induces necrosis of the host cell.
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the pathways by which ExoT alters the host cell cytoskeleton, (ii) the mechanism of Type III secretion dependent apoptosis, (iii)
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Based on this we developed a model for a novel error generating mechanism, and are now using recombinant Ty1 reverse transcriptase to study the biochemical basis for this phenomenon. Additionally, we have generated active site mutations in the Ty1 RT in the three conserved aspartates.
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We have been very successful in characterizing the molecular and biophysical defects caused by mutations in human voltage-gated sodium channels. Our studies utilize recombinant human sodium channels expressed heterologously in cultured cells and interrogated using electrophysiological tools such as the patch-clamp technique.
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Nuclear receptors are transcription factors that positively or negatively regulate gene expression in response to the binding of small molecular weight ligands. The human genome contains 48 members of this family that include receptors for steroid and thyroid hormones, vitamins, and metabolites of cholesterol and fatty acids.
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Department of Microbiology and Molecular Genetics University of California, Irvine We are studying the effects of mutations in the voltage-gated sodium channel to determine how specific alterations in channel function result in disease in the CNS, using three approaches. In the first part of our studies, we have examined the effects of a missense mutation in the mouse Scn8a gene encoding the Nav1.6 sodium channel, which is broadly distributed in brain and spinal cord.
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Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction.
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The resulting mapping and sequencing infrastructure provided a powerful foundation for us to then pursue complementary studies in human genetics, in particular those aiming to identify genes implicated in human disease. Projects within my laboratory led to the identification of the genes responsible for Pendred syndrome (a deafness/goiter disorder)
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The last round of vulval cell division, particularly the characteristic change of division axis of specific cells is considered an early landmark of vulval morphogenesis. Analysis of nhr-25 and lin-40 mutants indicated that they control the asymmetry of vulval cells and proper execution of the division pattern.
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D., Julie Jameson, Stephanie Rieder, and Richard Boismenu Department of Immunology The Scripps Research Institute The focus of my laboratory is to determine the antigen specificity and function of epithelial resident T cells. These cells appear to recognize and respond to self antigens expressed after malignancy, infection or trauma of neighboring epithelial cells.
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Division of Molecular and Human Genetics Children's Research Institute and Department of Pediatrics The Ohio State University My laboratory uses genetic approaches to try to understand the basis for selected inherited developmental disorders. Because many of these human disorders are extremely rare and genes on the X chromosome are generally conserved among all mammals, we have focused on X-linked disorders where we study a human disease using a mouse model that often has the same phenotype.
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We are currently further investigating the pathogenesis of the defects in affected male and female mice using techniques such as in situ hybridization and microarray expression analysis. We are also pursuing analyses of sterol trafficking and regulation in cultured cells derived from affected male embryos.
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Divisions of Human Biology and Clinical Research Fred Hutchinson Cancer Research Center The anti-apoptotic Bcl-2 family of proteins confers cellular resistance to a wide range of apoptotic triggers and multi-drug resistance for cancer cells. We developed a cell-based assay to screen for small molecule inhibitors of Bcl-xL, a closely related homolog of Bcl-2 with available NMR and crystallographically-determined solution structures.
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Non-peptidyl compounds related to antimycin may be clinically useful to target drug-resistant tumor cells overexpressing Bcl-xL. However, the inhibitory effect of antimycin on mitochondrial oxidative phosphorylation would preclude further drug development.
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A PROTEOME-WIDE SCREEN FOR PROTEINS REQUIRED FOR CANCER CELL INVASIVENESS BY HIGH-THROUGHPUT CALI Daniel Jay, Ph.D. Department of Physiology Tufts University School of Medicine A critical aspect of cancer is that cancer cells invade healthy tissue and metastasize to form secondary tumors.
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to use dyelabeled non-blocking antibodies and laser light to inactivate proteins in situ to validate their roles in cellular processes of clinical importance. We are now applying CALI to test which of the many proteins in the proteome is required for cancer cell invasiveness.
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Next, we found that the preference of the normal heart for fat versus carbohydrates as substrate for energy production is dynamically regulated during development and in diverse physiologic contexts. Importantly, in certain common acquired forms of heart disease, such as cardiac hypertrophy due to hypertension or coronary artery disease, the capacity of the myocardium to oxidize fats is dramatically reduced, a
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Pathologic cardiac hypertrophy or reduced oxygen availability, such as occurs with myocardial infarction or congenital heart disease, deactivates PPAR/PGC-1 at both transcriptional and post-transcriptional levels in rodents and humans. Conversely, the activity of PGC-1/PPAR, as a ligand-activated transcription factor, shows promise as a target for novel therapeutic strategies aimed at modulating myocardial metabolism in human disease states such as heart failure, diabetes, and myocardial infarction.
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We are currently identifying the genes responsible for evolutionary change within these regions, using many of the same forward-genetic and positional cloning methods we have previously used successfully to identify genes responsible for classical morphological traits in mice. These studies should make it possible to determine the number and type of molecular alterations that underlie evolutionary changes in natural populations.
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pathway, a receptor tyrosine kinase signaling pathway that plays a central role in blood vessel development in mammals, controls blood cell development in Drosophila. I will describe how the Drosophila VEGF pathway controls developmental migrations of blood cells, and how these and other recent results point to an intimate evolutionary and developmental association between blood cells and blood vessels.
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, are also significant contributors to the proliferation of breast and prostate cancer cells. These growth factor receptors and their signaling pathways as well as the coregulators associated with these nuclear receptors are excellent targets for therapeutics.
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SRA (steroid receptor RNA coactivator) , the only described RNA coactivator, plays an important role in this process, and is aberrantly expressed in human breast cancer cells.
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and amino acid (glutamate, aspartate, GABA, taurine) neurotransmitters in several regions of the human brain.
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However, this was not a consistent finding. In many cases seizures produced little or no perturbation of extracellular amino acid levels.
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The PTB domain in Shc allows it to bind to tyrosine phoshorylated growth factor receptors and connect certain growth factor receptors to the Ras signaling pathway. More recent work has shown that the PTB domains do not always bind to tyrosine phosphorylated proteins and can interact with proteins in a nonphosphotyrosine dependent fashion.
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We have found that Pals1 is targeted to the tight junction of epithelia cells by the L27N domain and that this domain binds a novel PDZ domain protein called Pals1 Associated Tight Junction Protein (PATJ)
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Protein separation is usually conducted by two-dimensional gel electrophoresis, which separates proteins based on size and charge. Identification of the gene encoding isolated proteins is done by mass spectrometry where one compares the
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, to rapidly identify protein changes between two or three samples on the same two-dimensional electrophoresis gel. This method relies on fluorescently tagging all proteins in each sample with one of a set of matched fluorescent dyes that do not affect the relative mobility of proteins during electrophoresis.
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I will describe our efforts at developing high-throughput screens based on RNAi methodology using Drosophila cell cultures in 384-well plates and automated microscopic imaging. In a pilot screen, we assembled a set of 1000 dsRNAs representing genes predicted to encode for regulators central to many fundamental cellular processes, including all the small GTPases and GTPase regulators, kinases, and phosphatases.
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We found that specific cathepsin B inhibitors blocked the processing of ROP2, suggesting that it was involved in rhoptry protein processing. When specific inhibitors were added to infecting parasites, invasion of host cells was blocked.
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Finally, we will look at the cellular localization of the putative hrp48regulated target RNAs in the RNA interference hrp48-disrupted cells using whole mount embryo in situ hybridization. Together, these molecular genetic analyses and the identification of putative hrp48- and PSI-regulated cellular target RNAs will help to understand the roles these RNA binding protein in play for Drosophila mRNAs.
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McKusick-Nathans Institute of Genetic Medicine The Johns Hopkins University School of Medicine Hypoxia-inducible factor 1 (HIF-1) is a global regulator of O2 homeostasis that activates the transcription of >50 known target genes encoding proteins which mediate cellular adaptation to O2 deprivation (via induction of glycolytic metabolism and growth factor signaling pathways)
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The Developmental Therapeutics Program at the NCI is presently screening for small molecule inhibitors of HIF-1 that can be utilized for proof-of-principle experiments in animal models and as lead compounds for the development of novel chemotherapeutic agents. Conclusion.
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The main approach that my laboratory has been taking to analyze the in vivo function of costimulatory pathways is to generate and analyze the immune capabilities of mice lacking costimulatory ligands and receptors using gene targeting approaches. We have focused primarily upon the B7:CD28 superfamily.
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Definition of the fundamental genetic abnormalities in cancer cells is a prerequisite to developing targeted therapies. Our work strongly argues that accurate animal models of human cancer are critical to this effort, as results obtained in cultured cells have
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The STI-571 program was nearly cancelled by Novartis less than 18 months before FDA approval due to the small perceived market opportunity, and resurrected only after direct pressure from leukemia patients. There are other leukemias with excellent tyrosine kinase targets for which drug development may never proceed due to the comparatively small number of patients with the disease.
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The requirement for Survivin for cell division explains why it is expressed in cancer cells, which are dividing, but not in most normal adult cells, which are not. A COMMON THEME IN THE PATHOGENESIS OF BACTERIAL INFECTION OF THE HUMAN RESPIRATORY TRACT Jeffrey N
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ChoP may function in binding and invasion of host cells through mimicry of the natural ligand for the receptor for platelet activating factor (rPAF) expressed on the apical surface of the respiratory epithelium.
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Several years ago, we discovered a novel transcription factor, FAST-1, that interacts in a ligand regulated manner with the TGFß signal transducers Smad2 and Smad4, and demonstrated that this interaction targets the Smads for specific, developmentally regulated promoters. The Smads are regulated directly by TGFß receptors, and therefore the identification of the Smad/FAST DNA binding complex provided the first example of a direct pathway by which TGFßs regulate a specific set of transcriptional responses.
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Using gene knockout technology, we generated mouse embryonic stem cells that lack the Ro protein. Mouse cells lacking Ro have drastically reduced levels of Y RNAs, suggesting that Ro protein binding stabilizes these RNAs from degradation.
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One differentially expressed gene encodes a small transcript in antisense orientation to a homolog of a negative regulatory protein kinase gene from another fungus, which is important in mating and starvation responses. We are examining both upstream and downstream aspects of this potential regulatory system in Hc, such as the specific environmental stimuli influencing expression of the antisense transcript, whether expression of the antisense transcript affects sense transcript expression, whether the sense transcript encodes a protein kinase functional in Hc, what the downstream targets of the putative kinase are, and the role of this locus in Hc biology and pathogenesis.
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We have particularly focused on growth factor/receptor systems that specifically act on a single or limited number of cell types, so that manipulation of these systems could be attempted so as to benefit diseases involving those cell types, without having widespread side effects. Over the last decade, our efforts have led to the discovery and characterization of multiple growth factor/receptor systems (e.g., neurotrophins and their Trk receptors; CNTF/IL6 family and their gp130-related receptors; agrin and its MuSK receptor; collagens and their DDR receptors; ephrins and their Eph receptors; angiopoietins and their Tie receptors;
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I will also discuss our new high-throughput knockout and transgenic technology, termed Velocigene, which we use to rapidly assign function to genes, with examples involving genes we have realized play key roles in the biology and pathology of muscle, cartilage, blood vessels, and lymphatic vessels.
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From page 152... ...
Given that 53BP1 form foci of similar size at sites of DNA damage and kinetochores, we speculate that 53BP1 provides functions that are shared by these two different cellular functions. Thus, our studies of the mitotic functions of 53BP1 may provide novel insights into the mechanism by which cells monitor and respond to double stranded DNA breaks.
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